The ability to isolate and concentrate rare cells or pathogens is fundamental to numerous biological and medical applications, often forming the starting point for down stream bioassays. Current approaches for cell isolation include centrifugation, filtration, fluorescent- and magnetic-activated cell sorting, and dielectrophoretic (DEP) cell separation. Among them, the DEP separator provides the advantages of speed, flexibility, controllability, and ease of application to automation. However, conventional DEP cell separators, containing two-dimensional (2-D) planar electrode arrays, often cannot process large volumes of samples at a time that is essential for rare cell or pathogen separation. A new three-dimensional (3-D) DEP cell/pathogen separation system is proposed, which is capable of processing large volume of samples with significantly improved collection efficiency. As examples of the relevance of the new system to clinical oncology, infectious disease and biowarfare, human monocytic (U937) and cervic carcinoma (HeLa) cells will be separated from blood, Escherichia coli and Salmonella typhimudum bacteria will be separated from blood, and Bacillus globigii will be concentrated from water. There examples address the needs of lowering the threshold for detection of cancer cells in blood, with the goal of diagnosing infectious disease and biowarfare agents without the need to first culture the pathogens.